Updated publication reference for PubMed record(s): 34210973. The PIKK superfamily member ATR is a key factor in DNA damage response (DDR) and is vital for the maintenance of genomic stability. DNA single strand breaks (SSBs) and replication stress activate ATR to phosphorylate a wide range of downstream substrates, which activates cell cycle checkpoint, senescence induction, cell death, and R-loop disintegration. ATR mutation causes the human ATR-Seckel syndrome, characterized by dwarfism, microcephaly and intellectual disabilities. Recent studies have implied ATR in non-nuclear functions; however, ATR's function in mitochondrial metabolism and a link to human diseases remains largely unknown. Here we show that ATR is located in mitochondria and its deletion alters mitochondrial dynamics prior to the DDR. ATR deletion disturbs the electron transfer chain (ETC) resulting in ROS overproduction and switches energy production from OXPHOS to the TCA cycle. Multi-omics analyses together with biochemical studies showed an imbalance of ETC proteins and membrane lipids accompanied with a dysregulation of key metabolic signaling pathways, including AMPK, mTOR and PGC1α. Pharmacological intervention of AMPK signaling or ETC functions delineates the metabolic pathways affected in ATR deleted cells. Mitochondrial metabolic dysfunction is more pronounced in ATR deleted neural cells and brain tissues, implicating a connection with neuropathological processes. Thus, ATR plays, beyond its well-known DDR function, an important role for cell metabolism and mitochondrial functionality, which contributes to the manifestation of neuronal deficit of ATR-Seckel.